This invention claims priority of the German patent application 100 45 873.4 which is incorporated by reference herein.
The present invention concerns a method for the analysis and evaluation of at least three-dimensional specimen data that are preferably detected with a confocal scanning microscope.
Methods of the generic type have been known for some time, and are used principally in the context of devices that can detect three- or multidimensional specimen data. Such devices can be, for example, computerized tomographs, magnetic resonance tomographs, or confocal scanning microscopes. With confocal scanning microscopes in particular, multi-dimensional specimen data can be detected. This can involve, for example, a time series of a detected three-dimensional specimen, i.e. the same specimen is detected three-dimensionally at respective predefined times, yielding a four-dimensional specimen data set (X, Y, Z, t). A further example of the existence of a multidimensional specimen data set is the detection of a three-dimensional fluorescent specimen using a confocal scanning microscope, in which context a separate detection channel can be provided for each fluorescent dye that is used. The detected specimen data set consequently has four dimensions (X, Y, Z, xcex).
The analysis and evaluation of multi-dimensional specimen data sets is problematic, however, in particular because of the large data volume. It is not readily possible to visualize specimen data sets whose dimension is greater than or equal to 3. A number of different visualization methods exist for this purpose. One cited purely by way of example is EP 0 908 849, which discloses a method and an apparatus with which, in a three-dimensional data set, a viewing point and a viewing direction proceeding from that viewing point can be defined. The three-dimensional specimen data set is then projected onto a plane that is perpendicular to the viewing direction, using the method of central projection with the viewing point as origin. The projected image is displayed on a monitor in the form of a pseudo-3D depiction.
The known analysis and evaluation methods are, however, problematic in many respects. Often the definition of the input values necessary for the respective methodxe2x80x94for example points, lines, or displacement directionsxe2x80x94needs to be performed by way of an interaction between user and computer. Often, however, this interaction requires extensive user training or a very considerable familiarization time, since following an interactive parameter input, the subsequent method steps often demand considerable processing time. If the result thereof does not meet expectations, another interactive input of the method parameters is necessary, after which the processing steps once again require processing time. For many applications, moreover, analysis and evaluation of the detected specimen does not require the utilization of a projection or some other complex processing method.
It is therefore the object of the present invention to describe and develop a method of the generic type with which, by unequivocal input of the necessary method parameters, it is possible to extract and output a portion of the detected specimen data, on the basis of which the three- or multi-dimensional specimen data can be analyzed and evaluated.
The method according to the present invention is achieved by the steps of:
detecting specimen data with a confocal scanning microscope and organizing the specimen data in a specimen data set,
defining at least two points of the specimen data set of the detected specimen data;
extracting at least one plane extending through the defined points from the specimen data set;
and graphically outputting the plane on an output unit.
Definition of the points preferably represents an input of the method parameters within the meaning of the object cited initially.
What has been recognized according to the present invention is firstly that the definition or input of points of a specimen data set is possible after only a very short training period. In particular, the accuracy of the definition of a point is greater than that of the user interfaces in which scroll bars or arrow buttons must be modified and the subsequent method steps are performed (in time-consuming fashion), and the result is output, only after a modification to the corresponding input means. It has furthermore been recognized that the output of a single plane or individual planes of the specimen data sets is often sufficient for the analysis and evaluation of multi-dimensional specimen data, so that complex process steps in the form of calculations and output steps can advantageously be omitted or at least minimized. The definition of at least two points of the specimen data set defines a family of planes that is output on an output unit. A storage medium, a monitor, and/or a printer/plotter is provided as the output unit. Output over a network, e.g. the Internet, is also conceivable. Output on a monitor or printer/plotter is performed graphically.
If a multi-dimensional specimen data set is present, coordinate values are determined or defined for all but three coordinates of the specimen data set. The result is to define a three-dimensional partial data set, provided for further processing, of the multi-dimensional specimen data set. The three coordinates whose values were not determined or defined span this partial data set. For example, a three-dimensional data set of a specimen could be acquired at each of twenty different points in time using a confocal scanning microscope. This four-dimensional specimen data setxe2x80x94comprising the coordinates X, Y, Z, and txe2x80x94would be reduced, by the definition of a specific time coordinate (e.g. the acquisition time corresponding to the tenth specimen data set), to a three-dimensional partial data set for further processing. The partial data set that is further processed is thus spanned by the three undetermined coordinates (X, Y, and Z).
In a first variant, provision is made for three points of the detected specimen data to be determined. This determination is accomplished in a three-dimensional specimen data set. Determination of the points could be accomplished by numerical input or by mouse clicking, i.e. very generally using means for marking or selecting points. An automatic determination would also be conceivable; for example, the three points could be the result of a segmentation process or a specimen recognition process.
Definition of the points is preferably accomplished on the basis of three mutually orthogonal section planes. These section planes visualize individual two-dimensional image sections through the three-dimensional specimen data set, in which context respective planes parallel to the XY plane, XZ plane, and YZ plane are usually displayed simultaneously on a monitor. The three section planes possess one common point, which can lie in the specimen to be analyzed. It would then be possible, by mouse clicking, to define in the displayed section planes three points for extraction of a plane from the specimen data set; it is not necessary to define a point in each displayed section plane.
The plane extending through three points is output on an output unit, i.e. for example on a monitor or color printer.
In a second variant, provision is made for only two points to be determined. This could be useful for analysis and evaluation of the detected specimen if the specimen has an elongated or cylindrical shape. The first point could accordingly be defined at one end of the specimen, and the second point at the other end of the specimen. The two defined points define a rotation axis. The planes that contain the rotation axis are then output. The planes are output in a sequence that forms a greater intersection angle each time with reference to the plane that was output first. For example, the plane that contains the rotation axis and e.g. the X axis could be output first. The next plane to be output could form an intersection angle of, for example, 5 degrees with the plane that was output first. The third plane to be output could form an intersection angle of 10 degrees with reference to the plane output first, so that the angle between the second and third planes is also 5 degrees. Very generally, provision is made for the intersection angle to increase by a constant value each time. The planes are output graphically, preferably on a monitor.
For the second variant, provision is made for the graphical output of the respective plane to be accomplished in a three-dimensional (3D) depiction of the detected specimen, displaying its spatial arrangement. In this context, provision is preferably made for a perspective 3D depiction that could be accomplished, for example, in the form of a vanishing-point depiction or in a parallel projection, i.e. a vanishing-point depiction in which the vanishing point is located at infinity. The rotation axis could also be plotted in the 3D depiction. In a preferred depiction, the detected specimen is depicted as being partially transparent, and the particular plane that is output is superimposed on the transparent depiction of the specimen. This advantageously clarifies the spatial relationship between the detected specimen and the plane being output, which considerably facilitates analysis of a complex three-dimensional specimen. Because of the 3D depiction of the planes being output, with this form of output only a small portion of the specimen is visible in many orientation directions of the plane. This is the case in particular in an orientation that is aligned substantially perpendicular to the entire depiction plane.
In the interest of flexible analysis and evaluation, provision is made hereinafter for defining, on the basis of a plane that has been output, the orientation and/or position of a further plane which is then output. A correction of the originally defined points can thereby be made immediately, i.e. the points do not need to be defined again in this stage of the method in another depiction form in order to extract the plane that is then desired. In the same fashion, the orientation and/or position of a previously defined rotation axis could be modified, resulting in output of the planes containing the new rotation axis. This procedure advantageously makes possible an optimization of the evaluation and analysis of the detected specimen, since further specimen regions of interest may have become detectable upon previous output of one or more planes. These regions can then be examined in more detail by directly defining once again the orientation and/or position of a further plane or a further rotation axis, for example directly in a perspective 3D depiction.
It would also be useful to output planes which are oriented parallel to a plane (preferably selected by the user) that has been output, and which each have an equidistant spacing. This output possibility corresponds to the usual output of sectional image planes, in which the planes that are output are oriented parallel to one of the coordinate planes.
In a specific output form, provision is made for graphical output of the planes to be accomplished two-dimensionally in each case. This could be accomplished in a two-dimensional rectangular region on a monitor; several two-dimensional rectangular regions could be output on a monitor in the form of a gallery, or each particular plane could be output on the entire monitor. The use of multiple monitors is also conceivable.
Very generally, the graphical output of the individual planes is accomplished in a time sequence. In this context, the speed profile of the time sequence could be defined by a user. An interactive request for output of the next plane by a userxe2x80x94for example by means of a keystroke or mouse clickxe2x80x94would also be conceivable in this context. For uniform output of the planes and thus an analysis of the specimen that is ergonomic for the user, provision is made for a substantially constant speed profile of the time sequence.
Prior to output of a plane, provision is made for a conversion to a common output grid of identical grid size and identical number of grid points. This common output grid is used for all planes that are output. The three-dimensional specimen data set generally exists in digital form, and an extraction of an individual plane is directly possible only if the extracted plane extends parallel to one of the three coordinate planes. As soon as the orientation of the plane being extracted deviates therefrom, however, it is necessary to extract from the specimen data set individual specimen points for which no directly detected values exist. The reason for this is the discrete digital grid that is defined by the imaging process of the confocal scanning microscope. A plane that has, for example, an intersection angle of 45 degrees with the XY plane, and that contains the X axis, would have along the X direction the same pixel spacing or grid spacing as the detected three-dimensional specimen data set. In the direction perpendicular thereto, however, the pixel spacing would have twice that value, since only there can a grid point of the detected three-dimensional specimen data set be extracted in each case. Depending on the orientation of the plane that is to be output, however, this would result in different output grids, making successful analysis and evaluation of a detected specimen difficult and, in some circumstances, even impossible.
An interpolation process, which for example could be implemented on the control computer of the scanning microscope, is accordingly provided for as the conversion for the purpose of output in a common output grid. When a measured value is to be extracted from the three-dimensional specimen data set at a location at which a measured grid point does not exist, a value that corresponds to the average of the adjacent measured values is therefore output at that location. The conversion could comprise a measured value adaptation that, in particular, compensates for physics-related imaging artifacts of the imaging process. One example of such an imaging artifact is bleaching of the fluorescent dyes. In this, in the context of a three-dimensional specimen detection, the measured fluorescent light intensity in the section plane detected first is, because of the bleaching behavior of the fluorescent dyes, greater than in the section plane detected last.
In very particularly advantageous fashion, provision is made for a plane that has been output to be re-imaged with the confocal scanning microscope. For this reimaging of the plane, the imaging parameters of the confocal scanning microscope are optimized. Depending on the orientation and position of the selected plane, the plane that has been output can, as a result of the interpolation process or the conversion to a common output grid, exhibit a spatial resolution that is far from optimum. Re-imaging of a plane could then be accomplished with an optimum spatial resolution accompanied by maximal noise suppression actions, for example by means of image averaging. Usually the plane is selected by the user before imaging.